Protocol for the identification and expression analysis of a cytoplasmic membrane-localized protein STING

Summary Here, we present a protocol for the detection of the two STING isoforms (erSTING and pmSTING) in human peripheral blood mononuclear cells or mouse splenocytes using Western blot and PCR. We detail steps to construct plasmids encoding each isoform and transfer them into mouse and human cell lines. Finally, we describe how to detect cell membrane localization of pmSTING using flow cytometry, immunoprecipitation, and immunofluorescence. This protocol is applicable for proteins with well-predicted topological structures. For complete details on the use and execution of this protocol, please refer to Li et al.1.


SUMMARY
Here, we present a protocol for the detection of the two STING isoforms (erST-ING and pmSTING) in human peripheral blood mononuclear cells or mouse splenocytes using Western blot and PCR. We detail steps to construct plasmids encoding each isoform and transfer them into mouse and human cell lines. Finally, we describe how to detect cell membrane localization of pmSTING using flow cytometry, immunoprecipitation, and immunofluorescence. This protocol is applicable for proteins with well-predicted topological structures. For complete details on the use and execution of this protocol, please refer to Li et al. 1 .

Institutional permissions
The protocol requires spleen tissue derived from C57BL/6J wild type (WT) mice and STING-deficient mice (C57BL/6J-Sting1 gt /J) and blood from healthy volunteers. Ethical approvals are required before starting this procedure. All animal experiments were carried out with the approval of the Harbin Medical University Research Ethics Committee.

Cell culture
Timing: 30 min

Isolation of human peripheral blood mononuclear cells (PBMCs)
Timing: 1-2 h Here, we describe how to isolate PBMCs from the peripheral blood of healthy volunteers.
1. Collect 3 mL blood into 5 mL EDTA anti-coagulation tubes and dilute with sterile PBS to 6 mL in a clean tube. 2. Then gently add the diluted 4 mL blood in the tube containing 4 mL Ficoll-Hypaque solution on the bottom. 3. Centrifuged the tube at 300 g, 30 min (troubleshooting 1). 4. After centrifugation, divide the solution in the tube into three layers, transfer the intermediate foggy layer to a blank tube and undergo another centrifugation at 300 g, 10 min. 5. Remove the RBCs by resuspending the cell precipitation in 1 mL RBC lysis buffer for 2 min. 6. Resuspend the cell precipitation with 5 mL PBS after centrifugation at 300 g ( Figure 1).
CRITICAL: These steps must be performed under sterile conditions.

Timing: 1-2 h
This part details how to isolate splenocytes from the spleens of C57BL/6J WT mice and C57BL/6J-Sting1 gt /J mice. 7. Sacrifice and immerse the mouse in 75% ethanol, then expose the spleen. 8. Harvest fresh spleens from WT or C57BL/6J-Sting1 gt /J mice and gently crush them in sterile PBS by the inner piston of the syringe. 9. Then suspend splenocytes in 10 mL PBS, filtrate them with the filter mesh (100 mm), and concentrate at 300 g, 10 min. 10. Remove the RBCs by resuspending cell precipitation in 1 mL RBC lysis buffer for 2 min. 11. After concentrating twice at 300 g, resuspended the cells with 5 mL PBS. 12. Resuscitate splenocytes by 4 mL RPMI 1640 and count for follow-up experiments (troubleshooting 2) ( Figure 2).
CRITICAL: After the mice were killed, the spleens were removed after disinfection with alcohol and operated under aseptic conditions.

OPEN ACCESS
This part details how to detect different STING isoforms in human PBMCs or C57BL/6 mouse splenocytes by WB.
13. Extract total proteins using RIPA buffer added with protease inhibitor PMSF and phosphatase inhibitors, and the final concentration of PMSF in protein solution is 0.2 mM. Add 100 mL precooled RIPA lysate per million cells. 14. Use a BCA Kit to quantify the proteins. 15. Separate equal amount of protein by electrophoresis and transfer onto a PVDF membrane, 50 mg protein loading for PBMCs and 40 mg protein loading for splenocytes. 16. Block membranes with 5% BSA at 25 C for 1 h. Incubated with anti-STING rabbit polyclonal antibody (19851-1-AP) diluted at 1: 600 in PBST and incubated for 14 h at 4 C.
Note: PVDF membranes can also be incubated with anti-STING rabbit polyclonal antibody (19851-1-AP) diluted at 1: 600 in PBST for 2 h at 25 C.
17. Wash the membranes three times by PBST 10 min each time, and incubate them with peroxidase-conjugated secondary antibodies (SA00001-1 and SA00001-2, diluted at 1: 2000 in PBST) for 1 h at 37 C.
Note: Increasing the number of washing times properly can decrease the background.
18. Wash the membranes three times by PBST for 10 min each time. Image with the ECL kit (KF003) and Bio-Rad Gel Doc XR + system.
Pause point: The proteins can be stored at À80 C for 6 months.

Timing: 2 h
This section details the detection of 2 distinct N-terminal STING isoforms in human PBMCs or C57BL/6 mouse splenocytes by PCR.
Pause point: Resulting RNA can be stored at À80 C for 1 month.

OPEN ACCESS
This section details how to detect the cytomembrane co-localization of STING isoform and classical membrane protein by immunofluorescent staining.
30. The C57BL/6J WT and C57BL/6J-Sting1 gt /J mice splenocytes smear samples preparation. a. Prepare clean polylysine-treated slides, blow the ten million cells evenly with 1 mL PBS, absorb the 30 mL suspension liquid with a pipette, drop it on the slide, let the liquid spread freely, form a monolayer cell structure, and let the cell suspension dry freely at 25 C. b. Use a hydrophobic barrier pen to circle the edge of the dry liquid surface to avoid fluid loss during subsequent operations. c. Use 2% cold paraformaldehyde (PFA) to fix the slides for 5 min and then use 1% BSA to block the slides in PBS for 30 min. Note: Gently wash the slides to retain more cells.

Timing: 7 days
This part details the method for erSTING and pmSTING plasmid construction.

Timing: 2 days
This part details how to perform immunoprecipitation to detect STING isoform expressed on the cytomembrane of human PBMCs or mouse splenocytes.

EXPECTED OUTCOMES
You can obtain information about mRNA and protein sequences of both human and mouse erST-ING 3,4 and pmSTING from the NCBI websites and their topological schematic from our previously published paper. 1 The STING isoforms detection results by WB and PCR are shown in our paper. 1 It clearly shows that there are two STING isoforms with different molecular weights in both mouse splenocytes and human PBMCs. The WB results show that the molecular weight of both mouse STING isoforms is 37 kD (erSTING) and 34 kD (pmSTING), respectively ( Figure 3B in ref. 1  The results of pmSTING detection with flow cytometry are shown in our paper. 1 Suppose a cytomembrane-localized STING isoform (pmSTING) exists in mouse splenocytes or human PBMCs. Flow cytometry results will show a shift of the staining of mouse pmSTING (Figure 2A in ref. 1 ) or human pmSTING ( Figure 5A in ref. 1 ) by using an antibody against the C-terminal epitope of STING compared to the control staining by using isotype or anti-GAPDH antibodies.
The immunofluorescence results of co-staining of pmSTING with cytomembrane protein markers of different immune cells in C57BL/6J WT or C57BL/6J-Sting1 gt /J mouse splenocytes are shown in our paper. 1 You can observe a strong co-localization between pmSTING and the cytomembrane protein markers (such as CD3, CD19 and CD11b) in non-permeabilized C57BL/6J WT splenocytes ( Figure 2C in ref. 1 ) in contrast, and you will fail to observe the co-localization between them in splenocytes of the C57BL/6J-Sting1 gt /J mouse ( Figure 2D in ref. 1 ). The immunoprecipitation results are also shown in our paper. 1 If there is a cytomembrane-localized isoform (pmSTING), the WB results will show the presence of IgG immunoblotting band only in the STING antibody immunoprecipitated lysates by using an anti-IgG antibody ( Figure 3E in ref. 1 ), after immunoprecipitation with the specific antibodies against STING C-terminal domain, against isotype or GAPDH.

QUANTIFICATION AND STATISTICAL ANALYSIS
For relevant analysis of cytomembrane-localized proteins, we recommend analyzing and presenting them in several different ways, such as immunofluorescence, co-localization, flow cytometry and immunoprecipitation, to ensure the accuracy of pmSTING localization and avoid the non-specific results of one experimental method.
For flow cytometry and immunofluorescence analysis, ensure that the number of cells, antibody dosage and concentration in each group were consistent.

LIMITATIONS
This method is unsuitable if the membrane protein is evenly spanned and the N or C terminus of the protein is located in the cytoplasm rather than toward extracellular, 5 and if it is difficult to obtain specific antibodies against the extracellular segment.
The spatial structure of the protein may mask specific epitopes, which may result in the use of a particular IP antibody, and the target protein complex will rarely be precipitated, no matter how much the antibody concentration is increased. Since the test is performed in the natural state, the proteins pulled down by IP may be different at different times and under different treatments, and of course, the accuracy of the protein obtained will become more and more significant as the number of experiments increases.

Potential solution
The diluted blood needs to be added gently to the upper layer of the ficoll in the centrifuge tube, it must be done gently to avoid mixing the two solutions, and eventually, it must be ensured that the two solutions are clearly layered. When separating PBMCs in the first step of centrifugation, note that the descending speed setting must be set to NO BREAK, or only 1-2% brake. Otherwise, the stratification will be confused.

Problem 2
The number of surviving splenocytes is too small.

Potential solution
During the grinding process, try to control the grinding force to ensure that the cell sieve is suspended and avoid mass cell death caused by direct grinding at the bottom of the dish. To ensure the vitality of cells, the whole operation process should be gentle to avoid causing mechanical damage to cells. To keep the cells in good condition, the whole experimental cycle should be shortened as much as possible.

Problem 3
The signals of cell surface markers are undetectable or inadequate.

Potential solution
Set up appropriate controls during flow cytometry analysis (refer to step-by-step method details 26): negative control to avoid false positive (anti-GAPDH), isotype control to eliminate background staining caused by non-specific binding of antibodies to the cell surface, positive control (if needed). In addition, be sure to add the secondary antibody at the recommended concentration and target the correct host of the primary antibody. If the membrane protein expression level is low, choose a bright fluorescent secondary antibody (such as PE) to detect it.

Problem 4
The stain was too strong or too weak during the immunofluorescence.

Potential solution
Following the experimental conditions of the antibody instructions, the antigen should be fully repaired and the epitope exposed to avoid false negative results. Select the appropriate antibody concentration, thoroughly wash off the excess antibodies, and avoid the broad background of the fluorescence image being too high.

Problem 5
More protein impurity bands precipitation.

Potential solution
Increase the concentration of salt ions in the washing buffer, appropriately increase the elution times, and reduce the incubation time. Pay attention to the molecular weight of the target protein.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Tongsen Zheng (zhengtongsen@hrbmu.edu.cn).

Materials availability
This study did not generate new unique reagents.

ll
OPEN ACCESS